Generation of transgenic mice (pronuclear injections)
Generation of chimeric mice (ES cell/blastocyst injections)
Cryopreservation of mouse embryos
Long term storage of cryopreserved embryos
Recovery of cryopreserved embryos
Rederivation of mouse strains to a specific pathogen free status
Derivation of mouse embryonic stem cells
Mouse tail vein injections
Blood sample collection
Timed matings
Generation of transgenic mice by
pronuclear injection
This technology is centered around the microinjection
of foreign DNA (transgenes) directly into the pronuclei of fertilized
zygotes. The DNA will randomly integrate into the genome of a
certain percentage of the injected embryos, resulting in mice that
now harbor this transgene and are capable of passing it on to future
generations.
Mice: For most transgenes, we use C57Bl/6 x DBA (F2) hybrid embryos. Upon request we can also put the transgene into other strains. We will inject and transfer a minimum of 300 embryos.
Three weeks after the birth of the potential founder transgenics, we will wean and take tail biopsies for the investigator for genotyping. Once the founder mice have been identified, they become the responsibility of the investigator. The investigator has the option to keep their mice in the barrier or can move them to a conventional colony elsewhere.
To get started, the investigator will need to provide us with 50 ug of restriction digested (to separate the plasmid sequences from the actual transgene) DNA along with a photo of a mini gel of the digest indicating which band is the transgene. The investigator will also have to have an IACUC approved animal use protocol number as well as provide billing information before the injections can begin.
Gene targeting in mouse embryonic stem cells by homologous recombination
In order to make a knockout or knockin mouse, the first step is to introduce the desired genetic mutation into pluripotent mouse embryonic stem cells. This can be accomplished by electroporating the vector DNA into ES cells and subsequent culturing the cells under selective conditions. The investigator will provide the facility with 25 ug of linearized targeting vector DNA in sterile water at a concentration of 1 ug/ul. We will electroporate and select with the appropriate drugs, pick and expand the drug-resistant clones, and ultimately freeze half and provide the other half to the investigator for genotyping. Correctly targeted clones will be thawed and expanded and refrozen for future use. We have germline competent ES cells in both the 129 and C57Bl/6 genetic backgrounds, as well as a newly derived hybrid ES cell line (129 x C57Bl/6) containing a tamoxifen inducible cre transgene.
Note: For both transgenic and gene targeting experiments, the design of the DNA construct to be used is critical- not only to achieve the desired experimental effect, but for accurate genotyping as well. We have several reference books on this and other mouse-related subjects that UT investigators are more than welcome to borrow.
Generation of chimeric mice by EScell/blastocyst injections
The primary purpose for creating chimeras is to
obtain germline transmission from the genetically manipulated ES cells. One
of the most common methods to accomplish this is the injection of the ES
cells directly into the cavity of blastocysts. Typically we will do three
days of injections (up to three different clones assuming three are available). If the ES
cells being used are those coming from our facility, all the investigator
needs to provide is an IACUC approved animal use protocol number and billing
information. If the cells to be injected are coming from an outside source,
the investigator must also provide credible documentation that they are free
of pathogens.
Cryopreservation of mouse embryos
There are three main reasons an investigator might want to cryopreserve genetically distinct mouse strains; (1) to prevent accidental loss of a valuable strain as a result of natural disaster (i.e. tornado, flood, power loss), human error, or disease, (2) as an alternative method to transport (as opposed to live mice), and (3) to take cages of mice not currently being used “off the shelf” and thereby avoid paying unnecessary per diems.
We will assist the investigator in generating large numbers of embryos from
super-ovulated female mice. Then we will collect the embryos and freeze
them in insemination straws.
Long term storage of cryopreserved embryos
To
maintain optimum viability, frozen embryos must remain in the liquid (not
vapor) phase of liquid nitrogen (LN2) until it is desired to thaw them. The
facility maintains an LN2 storage vessel specifically for this purpose.
Recovery of cryopreserved embryos
Upon request, we can thaw frozen embryos and implant them into recipient females. These embryos can be ones frozen and stored here as well as ones imported from an outside source.
Rederivation of mouse strains to a
specific pathogen free status
Please contact us to schedule and discuss
specific details.
Derivation of mouse embryonic stem cells
Using existing genetically altered mice as the starting material, we can isolate new embryonic stem cell lines harboring the desired mutation.
It never hurts to ask! If we can help you, we will.